The 26S proteasome complex is essential for targeted protein degradation. It is composed of two kinds of subcomplexes, namely a 20S core particle (CP) and a 19S regulatory particle (RP). The 20S CP confers the proteolytic activity to the proteasome, whereas the 19S RP which has six ATPase components shows ATP-dependence. Intriguingly, several recent studies have implicated the non-proteolytic role of the 19S RP in transcriptional activation independently of the 20S CP. However, the detailed regulatory mechanisms of transcriptional activation by the 19S RP remain largely unknown in vivo. In this grant application, we propose to determine the mechanism-of-action of the 19S RP in regulation of transcriptional activation in living eukaryotic cells, using yeast (Saccharomyces cerevisiae) as a model eukaryote. Our specific aims are -- (1) to determine the role of the 19S RP in establishing the specific protein interaction network at the promoter of a transcriptionally active gene in vivo, and (2) to determine the enzymatic roles of the 19S RP in formation of transcriptional initiation complex assembly (and hence transcriptional activation) in vivo. We will address these specific aims primarily using the FRET (fluorescence resonance energy transfer), ChIP (chromatin immunoprecipitation), mutational and transcriptional analyses. The outcomes of this research proposal will provide important information on the 19S RP regulation of eukaryotic transcriptional activation in vivo. Such knowledge will assist in the development of transcription-based therapeutic agents, since a growing number of human diseases are linked to aberrant transcriptional activation and/or are characterized by altered patterns of gene expression, and thus will have significant impact in public health. PUBLIC HEALTH RELEVANCE: Gene regulation is fundamental to the proper functioning of a cell, and many human diseases can be traced to abnormal gene regulation. In eukaryotes, gene regulation is largely controlled at the level of transcription which can be divided mechanistically into initiation, elongation and termination. Several proteins and multiprotein complexes, commonly known as "transcription factors", have been identified and characterized to regulate transcription both positively and negatively. Interestingly, a so-called non-transcription factor, namely the 26S proteasome complex that is essential for regulated protein degradation, has been implicated in controlling transcription at the levels of initiation, elongation and termination. However, the precise mechanisms by which the proteasome regulates transcription in living cells remain largely unknown. Understanding the regulatory mechanisms of gene expression by the proteasome components and their interactions with other intracellular proteins will be crucial for designing better therapeutic approaches for cancer and neurodegenerative diseases, since a growing body of evidence indicates the involvement of the proteasome in these diseases. Thus, our long-term research goal is to understand the mechanisms by which the proteasome regulates eukaryotic gene expression. In this application, we will focus our research on understanding the regulatory mechanisms of transcriptional initiation or activation by the proteasome.